Cystic fibrosis (CF) is the most common lethal, inherited disease in this country and the basic abnormality has not yet been elucidated. An aberrant fucosylation has been found in CF glycoproteins in many laboratories including our own. We have proposed an abnormal distribution of Alpha-L-fucosidase in CF based on an increased activity of the enzyme in CF skin fibroblasts and a decreased activity in serum. A defect in the biosynthesis and processing of Alpha-L-fucosidase could result in the abnormal distribution of this enzyme. A major thrust of this project will be to examine the biosynthesis of Alpha-L-fucosidase and follow the molecular forms of the enzyme in CF skin fibroblasts and culture medium and make a comparison to matched controls. For these studies we will purify the enzyme from CF fibroblasts and generate monoclonal antibodies to follow the formation and distribution of the radioactively labeled enzyme by immunoprecipitation and polyacrylamide gel electrophoresis. A membrane defect could produce many of the abnormalities described in CF. Endocytosis is a complex process by which macromolecules, including lysosomal enzymes, are transferred across cell membranes. Membrane recycling is postulated as a necessary step to maintain the required number of surface receptors. Preliminary studies, measuring the internalization of horseradish peroxidase showed that the CF fibroblasts had decreased endocytosis when compared to controls. In parallel studies, a glycoconjugate fraction was shown to be associated with endocytosis in hamster fibroblasts and we hypothesize that it is a fraction of the membrane which is recycled. To further understand the process of endocytosis we will characterize this glycoconjugate fraction and apply the information to the skin fibroblasts. We will use several different compounds which are inhibitors of endocytosis to study these phenomena. The synthesis and processing of Alpha-L-fucosidase has not been described and a precise role for fucose in biological systems remains to be defined. The proposed studies on cells from the genetic disorder CF, will provide information in these areas and could provide new insight into the pathogenesis of CF.